OBJECTIVE

This study was undertaken to investigate the effect of transdermal and oral estrogen replacement therapy in healthy postmenopausal women on markers of coagulation and fibrinolysis associated with coronary artery disease.

METHODS

In a randomized, placebo-controlled, double-blind study, healthy hysterectomized postmenopausal women received daily either placebo (n=49), transdermal <em>1</em>7beta-estradiol (E(<em>2</em>)) 50 microg (tE(<em>2</em>) group, n=33), oral E(<em>2</em>) <em>1</em> mg (oE(<em>2</em>) group, n=37), or oral E(<em>2</em>) <em>1</em> mg combined with gestodene <em>2</em>5 microg (oE(<em>2</em>)+G group, n=33) for thirteen <em>2</em>8-day treatment cycles. Hemostatic variables were measured in blood samples collected at baseline and in cycles 4 and <em>1</em>3.

RESULTS

No significant changes versus baseline and placebo were found in the tE(<em>2</em>) group, except for plasminogen activator inhibitor type-<em>1</em> (PAI-<em>1</em>) in cycle <em>1</em>3 (-3<em>2</em>.4%, P=.0<em>1</em>). In the oE(<em>2</em>) group, significant percentage changes from baseline versus placebo in cycle <em>1</em>3 were found in fibrinogen, -5.4% (P<.05); factor VII, -7.3% (P<.05); thrombin-antithrombin III complexes, -<em>1</em>3.3% (P<.05); tissue-type plasminogen activator (t-PA), -<em>1</em>7.3% (P<.00<em>1</em>); and PAI-<em>1</em>, -54.3% (P<.00<em>1</em>). In the oE(<em>2</em>)+G group, respective changes were factor VII, -<em>1</em>7.6% (P<.00<em>1</em>); t-PA, -<em>1</em>4.5% (P=.0<em>1</em>); PAI-<em>1</em>, -36.4% (P<.0<em>1</em>); and D-dimer, +<em>2</em><em>1</em>.8% (P<.05). No significant changes were observed in <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> and plasmin-alpha(<em>2</em>)-antiplasmin complexes.

CONCLUSIONS

Low-dose oral estradiol therapy was associated with an increase in fibrinolysis and small decreases in procoagulant variables. Transdermal therapy had minor effects.

The purpose of this study was to examine some aspects of umbilical cord blood collection for autologous transfusion in premature infants. All <em>1</em><em>2</em>0 microbacterial cultures (aerobic and anaerobic) of cord blood samples as well as 30 cultures of mycoplasma were treated. Cord <em>prothrombin</em> <em>fragment</em> (F <em>1</em> + <em>2</em>) concentrations were quantified at one and <em>1</em>0 minutes after clamping of the cord. F <em>1</em> + <em>2</em> concentrations assessed on <em>2</em>5 newborn infants were similar and no linear association with time of clamping could be drawn. This means that cord blood thrombosis is not activated for at least <em>1</em>0 minutes following clamping of the cord. As far as is known, the first newborn infant to benefit from this method of transfusion is reported here. The premature infant received two portions of autologous blood (on days 5 and 7). No untoward effects were noted. Blood, collected from the umbilical cord, is a safe source for autotransfusion, provided that bacteriological testing has been carried out.

BACKGROUND

Recent evidence suggests that regional left atrial coagulation activity may be increased in mitral stenosis and perhaps contribute to the pathophysiology of left atrial thrombus. However, the relation of left atrial coagulation activity to factors that predispose to left atrial thrombus formation is unknown, and the relation between left atrial and systemic coagulation activities is unresolved.

RESULTS

Left atrial and peripheral venous levels of the coagulation marker <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>) were measured in 3<em>2</em> patients with mitral stenosis with normal clotting times and no left atrial thrombus who were undergoing percutaneous balloon mitral valvuloplasty. Baseline peripheral venous F<em>1</em> + <em>2</em> levels, measured at the beginning of the valvuloplasty procedure, did not differ from those of 30 age-matched control patients. Prevalvuloplasty left atrial F<em>1</em> + <em>2</em> levels, obtained immediately after transseptal puncture, were similar to femoral venous levels in patients without left atrial spontaneous echo contrast (LASEC) (0.8<em>1</em> +/- 0.3<em>2</em> versus 0.8<em>1</em> +/- 0.<em>2</em>7 nmol/L, n = 7) but greater than femoral venous levels in patients with LASEC and either sinus rhythm (<em>1</em>.57 +/- 0.86 versus 0.99 +/- 0.38 nmol/L, n = <em>1</em>6, P < .00<em>1</em>) or atrial fibrillation (<em>1</em>.5<em>2</em> +/- 0.69 versus 0.85 +/- 0.33 nmol/L, n = 9, P < .003). Furthermore, LASEC emerged as the only significant predictor of increased regional left atrial coagulation activity (P = .005) on stepwise multivariate logistic regression analysis.

CONCLUSIONS

Increased regional left atrial coagulation activity in mitral stenosis occurs in the presence of LASEC, is evident in either sinus rhythm or atrial fibrillation, and is associated with normal systemic coagulation activity.

We performed a cross-sectional case-control study among <em>2</em>77 subjects with dementia and <em>2</em>98 control subjects drawn from participants of the Rotterdam Study, a population-based cohort study among subjects aged 55 years or over, and from participants of the Rotterdam Stroke Databank, a hospital-based stroke registry, with the objective to evaluate the association of indicators of coagulability, fibrinogen, <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em>, thrombin-antithrombin complex (TAT), and indicators of fibrinolysis, plasmin-inhibitor complex, D-dimer and tissue-type plasminogen activator (t-PA) with dementia. Increased levels of TAT, D-dimer and t-PA activity were associated with an increased risk of dementia. Additional stratified analyses indicated that an increased TAT level was the primary factor related to dementia. The present study provides evidence that predominantly increased thrombin generation is associated with dementia.

This report describes the successful use of protein C concentrate to treat severe purpura fulminans in a homozygous protein C-deficient infant for 8 months until oral anticoagulation was initiated. While fresh frozen plasma was previously used in such cases to replace protein C in the acute phase, the availability of a monoclonal antibody purified protein C concentrate now allows specific replacement of protein C, avoiding problems of fluid overload. An occlusive-hydrocolloid bandage proved to be effective in local treatment of skin lesions. D-dimer, fibrin monomer, thrombin-antithrombin complex and <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> were useful markers in monitoring and optimizing protein C replacement therapy.

CONCLUSIONS

Diagnosis of protein C deficiency should be considered in a newborn with purpura fulminans. Early diagnosis and adequate replacement therapy is life-saving. Today, administration of protein C is the acute as well as long-term therapy of choice.

OBJECTIVE

Stroke events occur most frequently in the morning hours. Impaired haemostatic activity and morning blood pressure (BP) surge, defined as the morning BP increase from sleep, have individually been associated with stroke risk in general or hypertensive populations. However, their combined impact on the risk of a stroke remains unknown.

RESULTS

A total of 5<em>1</em>4 hypertensive patients aged>> 50 years (mean 7<em>2</em>.3 years; 37% men) underwent <em>2</em>4 h BP monitoring, measurement of haemostatic risk factors [plasma fibrinogen, plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>), and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>(F<em>1</em>+<em>2</em>)], and brain MRI at baseline. The incidence of stroke was prospectively ascertained. During an average of 4<em>1</em> months (<em>1</em>75<em>1</em> person-years), there were 43 stroke events (ischaemic, 30; haemorrhagic, 5; undefined, 8). On multivariable analysis adjusted for confounding factors, the hazard ratio [HR (95% confidence interval (CI)] for stroke in the highest vs. lower quartiles of PAI-<em>1</em> was <em>2</em>.5 (<em>1</em>.3-4.6), that for F<em>1</em>+<em>2</em> was <em>2</em>.6 (<em>1</em>.4-5.0), and that for the morning BP surge was <em>1</em>.<em>2</em> (<em>1</em>.<em>1</em>-<em>1</em>.4; all P< 0.0<em>1</em>). In particular, the ratio was substantially higher in cases with the highest quartile of both PAI-<em>1</em> and F<em>1</em>+<em>2</em> levels compared with those with the lower quartiles of both parameters (HR: 8.<em>2</em>; 95% CI: 3.7-<em>1</em>8.<em>2</em>; P< 0.00<em>1</em>). Among the patients with the highest quartile of the morning BP surge (n= <em>1</em><em>2</em>8), the multivariable HR (95% CI) for the highest vs. lower quartiles of PAI-<em>1</em> was 3.4 (<em>1</em>.3-9.<em>1</em>) and that for F<em>1</em>+<em>2</em> was 3.3 (<em>1</em>.3-8.7) (both P< 0.05).

CONCLUSIONS

High levels of plasma PAI-<em>1</em> and F<em>1</em>+<em>2</em>, as well as an excessive morning BP surge, are independently and additively associated with an increased risk of stroke in older hypertensive patients.

The conversion of <em>prothrombin</em> to thrombin is catalyzed by <em>prothrombin</em>ase, an enzyme complex composed of the serine proteinase factor Xa and a cofactor protein, factor Va, assembled on membranes. Kinetic studies indicate that interactions with extended macromolecular recognition sites (exosites) rather than the active site of <em>prothrombin</em>ase are the principal determinants of binding affinity for substrate or product. We now provide a model-independent evaluation of such ideas by physical studies of the interaction of substrate derivatives and product with <em>prothrombin</em>ase. The enzyme complex was assembled using Xa modified with a fluorescent peptidyl chloromethyl ketone to irreversibly occlude the active site. Binding was inferred by prethrombin <em>2</em>-dependent perturbations in the fluorescence of Oregon Green(488) at the active site of <em>prothrombin</em>ase. Active site-independent binding was also unequivocally established by fluorescence resonance energy transfer between <em>2</em>,6-dansyl tethered to the active site of Xa and eosin tethered to the active sites of either thrombin or meizothrombin des <em>fragment</em> <em>1</em>. Comparable interprobe distances obtained from these measurements suggest that substrate and product interact equivalently with the enzyme. Competition established the ability of a range of substrate or product derivatives to bind in a mutually exclusive fashion to <em>prothrombin</em>ase. Equilibrium dissociation constants obtained for the active site-independent binding of <em>prothrombin</em>, prethrombin <em>2</em>, meizothrombin des <em>fragment</em> <em>1</em> and thrombin to <em>prothrombin</em>ase were comparable with their affinities inferred from kinetic studies using active enzyme. Our findings directly establish that binding affinity is principally determined by the exosite-mediated interaction of either the substrate, both possible intermediates, or product with <em>prothrombin</em>ase. A single type of exosite binding interaction evidently drives affinity and binding specificity through the stepwise reactions necessary for the two cleavage reactions of <em>prothrombin</em> activation and product release.

BACKGROUND

Left ventricular assist devices (LVADs) have provided a new therapeutic option for patients with end-stage heart failure. Despite advances in device design, there remains an apparent bleeding diathesis, which leads to increased transfusion requirements and reoperative rates. The purpose of our study was to examine the abnormalities that might contribute to these clinical sequelae.

RESULTS

To separate the effects of cardiopulmonary bypass (CPB), eight patients undergoing coronary revascularization (CABG) were compared with seven LVAD (TCI HeartMate) recipients intraoperatively and <em>2</em> hours postoperatively. We evaluated several well-characterized indexes of platelet activation: platelet count, platelet factor 4 (PF4), beta-thromboglobulin (beta-TG), and thromboxane B<em>2</em> (TXB<em>2</em>). We also measured activation of thrombin: thrombin-antithrombin III (TAT), <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), and fibrinopeptide A (FPA) as well as markers of fibrinolysis: plasmin-alpha <em>2</em>-antiplasmin (PAP) and D-dimer. Patterns of intraoperative platelet adhesion and activation were not statistically different in the CABG control and LVAD groups. In the immediate postoperative period, however, there was significant release of PF4 and beta-TG and generation of TXB<em>2</em>. Compared with the CABG controls (TAT, <em>2</em>6 +/- 8 micrograms/L; F<em>1</em> + <em>2</em>, 4 +/- <em>1</em> nmol/L; mean +/- SEM), there was a significant increase in TAT (380 +/- <em>1</em><em>1</em><em>2</em> micrograms/L) and F<em>1</em> + <em>2</em> (<em>2</em>3 +/- 4 nmol/L) in LVAD patients <em>2</em> hours after surgery. Furthermore, a sharp rise in FPA was noted <em>2</em>0 minutes after LVAD initiation (CABG, 8 +/- 4 ng/mL; LVAD, <em>2</em>35 +/- 63 ng/mL; P < .05). A concomitant increase in both PAP (CABG, 987 +/- <em>1</em><em>2</em>9 micrograms/L; LVAD 3456 +/- 7<em>2</em><em>1</em> micrograms/L; P < .05) and D-dimer (CABG, <em>1</em>678 +/- 4<em>1</em>6 ng/mL; LVAD, <em>1</em>5<em>2</em>43 +/- 468<em>2</em> ng/mL; P < .05) was observed.

CONCLUSIONS

The additive effects of CPB and LVAD lead to platelet activation as well as elevation of markers of in vivo thrombin generation, fibrinogen cleavage, and fibrinolytic activity. The etiology of these findings may be secondary to the LVAD surface, flow characteristics, and/or operative procedure. Nevertheless, platelet alterations and exaggerated activation of the coagulation and fibrinolytic systems may contribute to the clinically observed hemostatic defect.

BACKGROUND

There exists a current lack of information about the composition of the different types of plasma. No direct comparisons between apheresis plasma (AP) and recovered plasma (RP) derived from in-line-filtered whole blood (WB) have been published to date.

METHODS

Sixty AP units, <em>1</em>00 RP units from in-line-filtered WB held for 3 hours at <em>2</em>0 degrees C between donation and freezing, and an additional <em>1</em>00 RP units held for <em>1</em>5 hours at <em>2</em>0 degrees C before freezing were analyzed for coagulation factors and inhibitors, total protein, immunoglobulin G (IgG), and hemostasis and proteolysis activation markers. The influence of twice freezing and thawing on clotting factors V, VIII, and XI was also examined.

RESULTS

AP contains substantially greater activities of factor (F) V, FVIII, F IX, and FXI than RP frozen within 3 hours after WB donation. Prolonged holding of RP at <em>2</em>0 degrees C for more than <em>1</em>5 hours caused an additional reduction in FVIII, FXI, and protein S activities. Significantly greater levels of <em>prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em>, platelet factor 4, and neutrophil elastase were found in RP compared with AP. IgG was lower in AP compared with RP. Twice freezing and thawing caused a marked drop in FV, FVIII, and FXI activity.

CONCLUSIONS

Higher FVIII and F IX potencies in AP compared with RP can be expected to result in greater yields when used for purification of these clotting factors. AP is presumably more efficient than RP for treating coagulopathies. RP, however, may contain higher IgG levels than AP.

Forty-eight healthy pregnant women were studied prospectively and longitudinally. Blood sampling was performed at <em>1</em>0-<em>1</em>5, <em>2</em>3-<em>2</em>5, 3<em>2</em>-34 and 38-40 weeks of gestation, within one week and at eight weeks postpartum. Classic and modified activated protein C ratio decreased as pregnancy progressed. In the third trimester 9<em>2</em>% of the ratios measured with the classic test were above the lower reference level whereas all modified test ratios were normal. Slight activation of blood coagulation was shown with increased levels of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, soluble fibrin and D-dimer. Fibrinogen, factor VIII and plasminogen activator inhibitor type I and type <em>2</em> increased. Protein S and tissue plasminogen activator activity decreased. Protein C remained unchanged. No correlation was found between the decrease in classic APC ratio and changes in factor VIII, fibrinogen, protein S, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> or soluble fibrin, nor between the increase in soluble fibrin and changes in <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, fibrinogen and D-dimer.

Coagulation activation markers are being investigated as risk factors for cardiovascular disease; we examined the contribution of several lifestyle factors to variation in plasma levels of several activation markers measured in a population-based study of <em>1</em>947 men. Smoking, alcohol, body mass index (BMI), leisure and work activity, social class, and use of prescribed medicines were each examined in turn. Specific assays of fibrin D-dimer and von Willebrand factor (vWF) activity showed similar relationships to lifestyle variables as we observed previously for less specific assays of D-dimer and vWF antigen. D-dimer levels increased with age, in smokers and in men taking prescribed medication, and were negatively associated with leisure time activity. vWF activity increased with age and showed a U-shaped distribution with BMI. Factors VIIc and VIIIc and thrombin-antithrombin complexes were associated with BMI, factor VIIIc and <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>) were associated with age, activated partial thromboplastin time (aPTT) and F<em>1</em> + <em>2</em> were associated with smoking, and aPTT showed a small negative association with alcohol consumption. We conclude that lifestyle modification has the possibility of favourably influencing several of these risk markers. In particular, cigarette smoking has a possibly reversible effect on coagulation activation (measured by F<em>1</em> + <em>2</em> and D-dimer).

OBJECTIVE

The present study was conducted to evaluate the role of the duration of exercise and the impact of the exercise type for exercise-induced activation of coagulation.

METHODS

Eleven male triathletes were subjected to stepwise maximal (<em>1</em>7 min) and <em>1</em>-h maximal exercise in swimming, cycling, and running. Changes of hemostatic variable sand of plasma thrombomodulin, a marker of endothelial cell activation, were monitored.

RESULTS

Irrespective of the type of exercise, alterations in markers of thrombin (<em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, thrombin-antithrombin III complexes) and fibrin formation (fibrinopeptide A) were more pronounced after <em>1</em>-h exercise than after stepwise maximal exercise. Hemostatic parameters rose to the highest levels after running resulting in substantial fibrin formation as indicated by fibrinopeptide A increasing from <em>1</em>.33 ng.mL-<em>1</em> to <em>2</em>.<em>2</em>5 ng.mL (P < 0.05) after <em>1</em>-h exercise testing. Significant changes of plasma thrombomodulin were detected exclusively after running with increases from 38.<em>2</em> ng.mL-<em>1</em> to 44.<em>2</em> ng.mL-<em>1</em> (<em>1</em> h, P < 0.0<em>1</em>).

CONCLUSIONS

The data demonstrated that prolonged exercise is necessary for exercise-induced activation of coagulation resulting in thrombin and fibrin formation and suggested that endothelial cell activation possibly due to mechanical factors associated with running might play a role.

Human protein C is the precursor of a serine proteinase in plasma which contains nine 4-carboxyglutamic acid residues and functions as a potent anticoagulant. It is activated by thrombin in the presence of an essential endothelial-cell-membrane glycoprotein cofactor, thrombomodulin. In a purified human system, vitamin K-dependent proteins such as factor X, <em>prothrombin</em> and <em>prothrombin</em> <em>fragment</em> <em>1</em> were able to inhibit protein C activation by the thrombin-thrombomodulin complex, using either detergent-solubilized thrombomodulin or thrombomodulin reconstituted into vesicles consisting of phosphatidylcholine and phosphatidylserine (<em>1</em>:<em>1</em>, w/w). Factors VII and IX and protein S were much less efficient. <em>Prothrombin</em> <em>fragment</em> <em>1</em> behaved as a non-competitive inhibitor with apparent Ki values of 4 microM in the absence, and of <em>2</em>-<em>2</em>.5 microM in the presence, of phospholipids. Heat decarboxylation of <em>fragment</em> <em>1</em> abolished its ability to interfere in protein C activation, and high phospholipid concentrations could attenuate its inhibitory effect and were responsible for a gradual loss of the non-competitive character. <em>Fragment</em> <em>1</em> also inhibited the activation of 4-carboxyglutamic acid-domainless protein C, a proteolytic derivative of protein C lacking the 4-carboxyglutamic acid residues, without any influence from phospholipids. At high thrombin concentrations, with respect to thrombomodulin, the inhibitory effect of <em>fragment</em> <em>1</em> was diminished. <em>Fragment</em> <em>1</em>, at 3.8 microM, inhibited by 50% the activation of protein C (0.<em>1</em> or 0.3 microM) by thrombin. These results suggest that the 4-carboxyglutamic acid domain of vitamin K-dependent proteins can act as a modulator of the protein C anticoagulant pathway through two distinct types of interaction. The functional 4-carboxyglutamic acid domain would be necessary to allow the enhancement of protein C activation in the presence of anionic phospholipids and it could recognize a phospholipid-independent binding site on the thrombin-thrombomodulin complex.

Several studies have previously reported high levels of total tissue factor pathway inhibitor (TFPI) antigen in patients with hypercholesterolemia. The relationship between serum lipid concentrations and total and free-form TFPI antigen in 3<em>2</em> patients with primary type II hypercholesterolemia and 38 age- and gender-matched normolipemic control subjects was studied (Study Group I). Plasma concentrations of total TFPI (tTFPI) antigen, free-form TFPI (fTFPI) antigen, tissue factor antigen, factor VII activity (FVIIc), and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) were measured. The median levels of tTFPI, fTFPI, FVIIc, and F<em>1</em>+<em>2</em> were higher in hyperlipidemic patients compared with those in healthy subjects. The effect of lowering total cholesterol on hypercoagulability in <em>2</em>5 patients with type II hyperlipoproteinemia (Study Group II) were also studied. The median levels of tTFPI, FVIIc, and F<em>1</em>+<em>2</em> decreased significantly after 6 months of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor therapy in the hypercholesterolemic patients. On the other hand, fTFPI did not change after therapy. Plasma tTFPI was strongly correlated with total cholesterol and low density lipoprotein (LDL)-cholesterol in hyperlipidemic patients. In contrast to the strong correlation between tTFPI and total cholesterol, the correlation between plasma fTFPI and total cholesterol was relatively poor. These results suggest that the activation of the anticoagulant system as well as the activation of the coagulation system may occur in association with hypercholesterolemia. Furthermore, the results of this study may suggest that lowering of total cholesterol in hyperlipidemic patients reduces the thrombin generation in plasma and that down-regulation of LDL does not affect the anticoagulant potency of TFPI in plasma.

Erythromelalgia, a characteristic aspirin-responsive microvascular thrombotic complication in essential thrombocythemia (ET), may develop despite oral anticoagulant treatment or treatment with heparin, suggesting that the generation of thrombin is not a prerequisite for its development. To study this, a cross-sectional comparison of the plasma levels of thrombomodulin (TM), platelet factor 4 (PF4), beta-thromboglobulin (beta-TG), <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>) and total degradation products of fibrin(ogen) (TDP) was made between 5 ET patients suffering from erythromelalgia, <em>1</em>6 asymptomatic ET patients and <em>2</em>0 control subjects, and after treatment with aspirin, respectively. Furthermore, <em>2</em> ET patients with a history of erythromelalgia were studied at regular time intervals after discontinuation of aspirin until erythromelalgia recurred. As compared with asymptomatic ET patients and control subjects erythromelalgia was characterized by significantly higher beta-TG and TM levels but no significant differences were detected in either F<em>1</em> + <em>2</em> or TDP levels. Treatment of erythromelalgia with aspirin resulted in disappearance of erythromelalgic signs and symptoms, which was paralleled by a significant decrease of beta-TG and TM levels. Histopathologic and immunohistochemical analysis of biopsies derived from erythromelalgic skin areas of <em>2</em> ET patients showed that erythromelalgic thrombi stained positively for von Willebrand factor opposed to only a weak fibrin staining. Our data suggest that erythromelalgia is caused by the intravascular activation and aggregation of platelets with subsequent sludging or occlusion of the acral arterial microvasculature. The generation of thrombin appears not to be essential for the formation of these platelet thrombi, thereby giving a plausible explanation for the inefficacy of coumadin derivatives and heparin in the prevention and treatment of erythromelalgia in essential thrombocythemia.

BACKGROUND

Edoxaban, a direct factor Xa inhibitor, is a once-daily, non-vitamin K antagonist oral anticoagulant. There is no established method to reverse the activity of non-vitamin K oral anticoagulants in cases of hemorrhage or urgent surgery. This study evaluated the ability of a 3-factor prothrombin complex concentrate (3F-PCC) to reverse the anticoagulatory effects of edoxaban.

METHODS

In this phase 1 study, 24 healthy subjects were randomly assigned to receive a single dose of 60 or 180mg edoxaban, followed by placebo, 25IU/kg 3F-PCC, or 50IU/kg 3F-PCC. Edoxaban pharmacokinetics and pharmacodynamics, including the primary endpoint of prothrombin time (PT) and endogenous thrombin potential (ETP), were assessed. D-dimer and prothrombin fragment 1 and 2 (F1+2) were also measured.

RESULTS

Overall, there were no apparent consistent effects of 3F-PCC on edoxaban pharmacokinetics. Administration of 3F-PCC 25 or 50IU/kg with edoxaban 60 or 180mg did not substantially accelerate the return of PT to baseline levels. However, infusion of 3F-PCC 25 and 50IU/kg did substantially accelerate return to baseline of ETP compared with placebo. D-dimer and F1+2 data did not indicate any lasting procoagulant effects of 3F-PCC infusion, although a transient increase in F1+2 was noted during and after 3F-PCC infusion. Edoxaban and 3F-PCC co-administration was well tolerated in normal healthy subjects.

CONCLUSIONS

There was no apparent reversal of PT prolongation with 3F-PCC following edoxaban infusion, but ETP was completely reversed. Co-administration of 3F-PCC was well tolerated, but a dose-dependent increase in F1+2 may reflect a procoagulant risk.

Systemic activation of coagulation leading to disseminated intra-vascular coagulation (DIC) is an important feature in patients with severe sepsis. Tissue factor has been shown to play a primary role in this pathological response, as revealed by the use of specific inhibitors and antagonists of the tissue factor/factor VIIa pathway. This class of agents has been demonstrated to attenuate the coagulation response in human volunteers with induced low-grade endotoxemia and to reduce mortality in primate models of Gram-negative sepsis. The efficacy of these agents in attenuating the activation of coagulation and formation of microvascular thrombosis in sepsis may depend on the mechanism of inhibition. Here we demonstrate the efficacy of recombinant nematode anticoagulant protein c<em>2</em> (rNAPc<em>2</em>) that specifically inhibits the tissue factor/factor VIIa complex by a novel mechanism, in a model of endotoxin-induced coagulation activation in chimpanzees. Administration of a low dose of Gram-negative endotoxin induced marked increases of thrombin generation as measured by plasma levels of <em>prothrombin</em> activation <em>fragment</em> F(<em>1</em>+<em>2</em>) and thrombin-antithrombin complexes, which were completely blocked by rNAPc<em>2</em>. In chimpanzees receiving rNAPc<em>2</em> alone, there was a significant reduction in the activation of factor X but not factor IX, compared to animals receiving placebo. In contrast to the effect of rNAPc<em>2</em> on thrombin generation, there was no effect of this inhibitor on the well known enhanced systemic fibrinolytic response induced by endotoxin. In conclusion, the recombinant peptide rNAPc<em>2</em> is an effective inhibitor of tissue factor-driven thrombin generation during low grade endotoxemia. These results suggest that rNAPc<em>2</em> may be a promising therapeutic option to inhibit coagulation activation in patients with sepsis.

Anticoagulants have gained increasing attention in the treatment of sepsis. This study used danaparoid to investigate the role of factor Xa in endotoxin-induced coagulation and inflammation and its effectiveness when coagulation activation has already occurred. Thirty healthy volunteers were enrolled in the randomized, placebo-controlled trial. Subjects received <em>2</em> ng/kg endotoxin and danaparoid <em>1</em>0 min or 3 h thereafter or placebo. Endotoxin increased <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F(<em>1</em>+<em>2</em>)) levels from 0.5 to 7.0 nmol/L at 5 h in the placebo group. Early danaparoid infusion inhibited endotoxin-induced thrombin formation: maximum F(<em>1</em>+<em>2</em>) levels reached only <em>1</em>.8 nmol/L (P<.0<em>1</em>, vs. baseline or placebo). Delayed danaparoid infusion effectively blocked further thrombin formation. However, danaparoid did not alter endotoxin-induced changes in the fibrinolytic system, cytokine levels, activation of leukocytes, or tissue factor expression on monocytes. Danaparoid therefore selectively attenuates endotoxin-induced coagulopathy, even with delayed administration when coagulation activation is well under way.

The effects of two oral contraceptives, containing gestodene and either <em>2</em>0 micrograms or 30 micrograms ethinylestradiol, on hemostatic parameters was investigated in a six-month randomized study involving a total of 40 healthy women between the ages of <em>1</em>8 and 30 years. A large number of hemostatic parameters were measured, which were categorized as either pro-coagulatory, anti-coagulatory, profibrinolytic, anti-fibrinolytic or indicative of fibrin turnover. Additionally, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-<em>1</em>) were measured before and after venous occlusion and delta and ratio values calculated. Pro-coagulatory factors as well as reaction products reflecting in vivo coagulatory activity (thrombin-antithrombin III complex, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>) were found to increase. Among the anti-coagulatory parameters, only protein S concentration and protein S activity decreased, most notably in the 30 micrograms EE group. There was a corresponding increase in fibrinolytic activity reflected by reaction products of in vivo fibrinolysis (plasmin-antiplasmin <em>2</em>-complex, fibrin-degradation products). Measurement of t-PA and PAI-<em>1</em>, before and after venous occlusion, revealed that the fibrinolytic response was more pronounced in the <em>2</em>0 micrograms EE group. There was also an increase in the threshold of fibrinolytic inhibition (ratio PAI-<em>1</em>) in both groups, which was less pronounced in the <em>2</em>0 micrograms EE group. Apart from isolated measurements, all parameters remained within their normal ranges and values returned to baseline in the follow-up cycle. It is concluded that both preparations had a balanced effect on the hemostatic system stimulating both pro-coagulant and fibrinolytic activity. No statistically significant differences were observed between the two groups; however, there was a trend towards greater fibrinolytic capacity in the <em>2</em>0 micrograms EE group.

BACKGROUND

The prothrombotic, hypofibrinolytic state that develops in patients with intermittent claudication (IC) upon walking due to ischemia-reperfusion injury (IRI) of the leg muscles may contribute to the high incidence of life- and limb-threatening thrombotic events observed in this patient group. Treatments, such as angioplasty, that obtund the IRI also ameliorate the procoagulant diathesis. The effect on this diathesis of supervised exercise and cilostazol, both of which provide symptomatic benefit in IC, but without significantly obtunding IRI, is unknown.

METHODS

Thirty-four patients (<em>2</em>7 men and 7 women; median age, 67 years; range, 63-7<em>2</em> years) were randomized to receive best medical therapy (BMT) plus supervised exercise (n = 9), BMT plus cilostazol (n = 9), BMT plus supervised exercise plus cilostazol (n = 7), or BMT alone (n = 9) in a <em>2</em> x <em>2</em> factorial design. Thrombin-antithrombin complex and <em>prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em>, both markers of thrombin generation; plasminogen activator inhibitor antigen and tissue plasminogen activator antigen, both markers of fibrinolysis; ankle-brachial pressure index (ABPI); and initial and absolute claudication distance (ACD) were measured at baseline and then 3 and 6 months after randomization.

RESULTS

At 6 months, when compared with receiving BMT only, supervised exercise and cilostazol resulted in improvements in ABPI of <em>1</em>8% and <em>1</em>3% and in ACD of 40% and 64%, respectively. The effects on ABPI and ACD of combining supervised exercise and cilostazol were additive. Supervised exercise, cilostazol, and supervised exercise combined with cilostazol had no significant effect on any of the four hemostatic markers.

CONCLUSIONS

Treatment of IC by supervised exercise or cilostazol results in significant improvements in ABPI and ACD but has no demonstrable effect on the prothrombotic diathesis. This suggests that supervised exercise and cilostazol, unlike angioplasty, are unlikely to have a long-term beneficial effect on the thrombotic risks faced by these patients.

Low-molecular-weight and unfractionated heparins are frequently used to treat venous thromboembolism, but it is not known whether they are equally effective in inhibiting in vivo generation of thrombin. In this multicenter trial, <em>1</em>048 patients were randomized to intravenous unfractionated heparin (group A), twice daily low-molecular-weight heparin (reviparin) for <em>1</em> week (group B), or once daily reviparin for 4 weeks (group C). All patients received vitamin K antagonists. Blood samples withdrawn at the baseline and at weeks <em>1</em> and 3 were analyzed using markers of in vivo thrombin generation and other coagulation parameters. During the first 3 weeks symptomatic recurrent deep vein thrombosis-pulmonary embolism (DVT/PE) occurred in <em>1</em>7 (4.5%) of 375 patients in group A compared with 4 (<em>1</em>.0%) of 388 patients in group B, and 9 (<em>2</em>.4%) of 374 patients in group C. Forty percent of patients in group A, 53.4% in group B, and 53.5% in group C showed 30% or greater reduction in thrombus size assessed by venography. Patients in group B had significantly greater reduction in D-dimer, <em>prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em> (F<em>1</em> + <em>2</em>), endogenous thrombin potential (ETP), and thrombin-antithrombin (TAT) complexes compared to groups A and C. Greater release of tissue factor pathway inhibitor (TFPI) and reduction in levels of thrombin activatable fibrinolysis inhibitor (TAFI) and fibrinogen were significantly more pronounced in group C patients. Reviparin administered twice daily plus vitamin K antagonist is more effective in inhibiting in vivo thrombin generation compared to intravenous unfractionated heparin plus vitamin K antagonist, and reviparin once daily produced significantly higher TFPI release and greater reduction in TAFI and fibrinogen levels.

BACKGROUND

Recombinant hirudin, a pure, specific antithrombin could be more effective than heparin in the treatment of deep vein thrombosis, but its short half-life requires constant intravenous infusion, whereas subcutaneous administration of recombinant hirudin can ensure stable and prolonged plasma levels. The aim of our study was to assess the pharmacokinetics, the results on the coagulation variables, and the safety of a recombinant hirudin (HBW 0<em>2</em>3) administered subcutaneously in patients suffering from deep vein thrombosis.

METHODS

Recombinant hirudin (HBW 0<em>2</em>3) was administered subcutaneously to <em>1</em>0 patients with recent deep vein thrombosis, at a dose of 0.75 mg/kg of body weight twice daily for 5 days, after which standard heparin and acenocoumarol were introduced. Bilateral lower limb venography, and pulmonary angiography, and/or ventilation-perfusion lung scan were carried out on day <em>1</em> prior to recombinant hirudin injection and repeated on day 5. aPTT and recombinant hirudin plasma levels were serially assessed after the <em>1</em>st and the <em>1</em>0th injections. <em>Prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>, thrombin-antithrombin III complexes, fibrin degradation products were collected on days <em>1</em> and 5.

RESULTS

Clinical evolution was uneventful in all but one patient who had a probable recurrence of pulmonary embolism on day 4. No hemorrhagic complication, no untoward biological event was observed. On days 5, Marder score was unchanged or had decreased. Plasma levels of recombinant hirudin peaked in between 3 and 4 h following the injection. aPTT values paralleled, and were significantly correlated with plasma levels of recombinant hirudin on day <em>1</em> as well on day 5 (r = 0.903, r = 0.948 respectively). Fragment <em>1</em> + <em>2</em>, and thrombin antithrombin complexes non-significantly decreased from day <em>1</em> to day 5.

CONCLUSIONS

Subcutaneous administration of recombinant hirudin ensures prolonged stable plasma levels of recombinant hirudin which results in efficient anticoagulation. A dose-ranging study conducted with subcutaneous recombinant hirudin in comparison to conventional heparin therapy may answer the question as to efficacy.

Activation of <em>prothrombin</em> and the subsequent reactions of thrombin with its substrates and its major inhibitors, antithrombin III (AT III) and heparin cofactor II (HC II), likely reflect both intravascular and extravascular coagulation. Several studies have reported increased in vivo coagulation in cancer. Whether the increased thrombin production in malignancy is accompanied by a corresponding increase in thrombin inhibition is unknown. This study quantified <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), thrombin-AT III (TAT), thrombin-AT III-vitronectin (TAT.V), and thrombin-HC II-vitronectin (THCII.V) in the plasmas of healthy volunteers (n = 37); patients with localized solid tumours before treatment was initiated (n = 39); and five patients with non-Hodgkin's lymphoma, both before and during weekly chemotherapy. Two of the five non-Hodgkin's lymphoma patients developed deep venous thrombosis (DVT) during chemotherapy. In normal plasma, where the concentrations of the four parameters likely reflect haemostasis, the sum of TAT, TAT.V and THCII.V was 6<em>1</em>% that of F<em>1</em> + <em>2</em>, compared with 30% in cancer plasmas. In addition, the mean +/- SEM of F<em>1</em> + <em>2</em> in the plasmas of cancer patients (<em>1</em>.56 +/- 0.09 nM) was significantly elevated (P < 0.00<em>1</em>) when compared with healthy volunteers (0.89 +/- 0.06 nM). Eight weeks of chemotherapy increased the F<em>1</em> + <em>2</em> and the binary TAT in plasmas of the non-Hodgkin's lymphoma patients by approximately <em>1</em>.5- and <em>2</em>.9-fold, respectively. Thus, increased <em>prothrombin</em> activation in cancer patients, without corresponding increases in concentrations of thrombin-inhibitor complexes, raise the possibility that a significant portion of the thrombin generated in vivo escapes inhibition in cancer and contributes to the high risk of DVT in malignancy.

BACKGROUND

Diagnosis of acute coronary syndromes (ACS) is a major challenge for emergency physicians. Because soluble fibrin (sF) has been suggested as a potential early marker of impending myocardial ischemia, we were interested whether a sF bedside test could help in early identification of patients with ACS in the emergency department.

METHODS

We evaluated plasma coagulation markers, including a newly developed sF bedside test, <em>prothrombin</em> <em>fragment</em> (F(<em>1</em>+<em>2</em>)), sF, and D-dimer, in a cross-sectional trial with <em>1</em>84 patients suggestive of ACS.

RESULTS

Whereas 76% (<em>1</em>3 of <em>1</em>7) of patients with unstable angina pectoris (UAP) had a positive sF bedside test, only <em>1</em>0 of 33 patients (30%) with non-ST-segment-elevation myocardial infarction and <em>1</em>0 of 44 patients (<em>2</em>3%) with ST-elevation myocardial infarction tested positive. Three percent of controls (<em>1</em> of 33) and <em>1</em><em>1</em>% of patients (6 of 57) with preexisting stable angina had a positive sF bedside test (P <0.00<em>1</em> for noncardiac chest pain vs ACS), yielding an overall specificity of 9<em>2</em>% and a sensitivity of 35%. The sensitivity of the established coagulation markers was significantly less to detect ACS (<em>1</em><em>1</em>% for F(<em>1</em>+<em>2</em>), <em>2</em>0% for thrombus precursor protein, and <em>1</em>8% for D-dimer; P <0.0<em>2</em> vs sF bedside test). The sF bedside test presented the earliest objective indicator of impending myocardial damage in the majority (<em>1</em>0 of <em>1</em>3) of ACS patients with a normal or nondiagnostic electrocardiogram (ECG).

CONCLUSIONS

A sF bedside test offers a specific tool for early identification of patients with ACS in an emergency department setting, although its sensitivity seems sufficient only for the early identification of patients with UAP. A sF bedside test could be useful, particularly in UAP patients with a nondiagnostic ECG.